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European	Journal	of	Chemistry	5	(1)	(2014)	101‐110	

	

European	Journal	of	Chemistry	
ISSN	2153‐2249	(Print)	/	ISSN	2153‐2257	(Online)		2014	Eurjchem	Publishing	‐	Printed	in	the	USA	

http://dx.doi.org/10.5155/eurjchem.5.1.101‐110.882	

	
	

	

	

European	Journal	of	Chemistry	
Journal	homepage:	www.eurjchem.com	

	 	 	

A	simple	and	rapid	spectrophotometric	method	for	the	determination	of	iron		
in	environmental,	biological,	pharmaceutical,	food	and	soil	samples	using		
1,2‐dihydroxybenzene‐3,5‐disulfonic	acid	

Tasnima	Zannat	and	Mohammed	Jamaluddin	Ahmed	*	

Laboratory	of	Analytical	Chemistry,	Department	of	Chemistry,	University	of	Chittagong,	Chittagong,	4331,	Bangladesh	

*Corresponding	author	at:	Laboratory	of	Analytical	Chemistry,	Department	of	Chemistry,	University	of	Chittagong,	Chittagong,	4331,	Bangladesh.		
Tel.:	+88.031.618236.	Fax:	+88.031.2606014.	E‐mail	address:	pmjahmed55@gmail.com	(M.J.	Ahmed).	

	
	

	 	

	 	 	
ARTICLE	INFORMATION	 	 ABSTRACT	

	
DOI:	10.5155/eurjchem.5.1.101‐110.882	

Received:	18	July	2013	
Received	in	revised	form:	07	September	2013	
Accepted:	07	September	2013	
Online:	31	March	2014	

KEYWORDS	

	 An	 ultra‐sensitive	 and	 highly	 selective	 non‐extractive	 spectrophotometric	 method	 is
presented	for	the	rapid	determination	of	iron	(III)	at	trace	levels	using	1,2‐dihydroxybenzene‐
3,5‐disulfonic	 acid	 (Tiron)	 as	 a	 new	spectrophotometric	 reagent	 (max	=	 665	nm)	 in	 slightly
acidic	 aqueous	 (2×10‐6	 ‐	 2×10‐5	 mol/L	 H2SO4)	 solution.	 The	 reaction	 is	 instantaneous	 and
absorbance	 remains	 stable	 for	 over	 24	 h.	 The	 average	 molar	 absorption	 coefficient	 and
Sandell’s	sensitivity	and	detection	limit	were	found	to	be	6.0×105	L/mol.cm,	10	ng/cm2,	and	1
µg/L,	 respectively.	 Linear	 calibration	 graphs	 were	 obtained	 for	 0.02‐70.00	mg/L	 of	 Fe(III).
The	stoichiometric	composition	of	the	chelate	is	2:3	(Fe(III):Tiron).	Large	excesses	of	over	50
cations,	anions	and	complexing	agents	(e.g.	Tartrate,	oxalate,	citrate,	phosphate,	thiourea,	and
thiocyanate)	do	not	interfere	in	the	determination.	The	method	was	successfully	used	in	the
determination	of	iron	in	several	standard	reference	materials	(Alloys	and	steels)	as	well	as	in
some	 environmental	waters	 (portable	 and	 polluted),	 biological	 samples	 (Human	 blood	 and
urine),	food,	pharmaceutical	and	soil	samples,	solution	containing	iron	(II)	and	iron	(III)	and
some	complex	synthetic	mixtures.	The	method	has	high	precision	and	accuracy	(s	=	±0.01	for
0.5	mg/L).	

Tiron	
Biological	samples	
Iron	determination	
Spectrophotometry	
Environmental	samples	
Pharmaceutical	samples	

	
1.	Introduction	
	

Iron	 plays	 a	 dual	 role	 in	 human	 biochemistry	 as	 in	 trace	
amounts,	 it	 is	 an	 essential	 nutrient,	 while	 large	 amounts	 are	
toxic	and	carcinogenic	[1].	The	essentiality	and	toxicity	of	iron	
depend	 on	 its	 oxidation	 states	 or	 the	 forms	 in	 which	 it	 was	
supplied.	Iron	in	trace	amounts	is	important	industrially	[2],	as	
a	 biological	 nutrient	 [3],	 toxicant	 [4],	 environmental	pollutant	
[5],	 and	 occupational	 hazards	 [6].	 The	 industrial	 uses	 of	 iron	
and	 its	 compound	 are	 too	 numerous	 [7].	 It	 is	 the	 major	
constituent	in	steel	making.	Several	iron	oxide	form	find	use	as	
paint	 pigments,	 polishing	 compounds,	 magnetic	 inks,	 and	
coatings	 for	 magnetic	 tapes.	 The	 soluble	 salts	 are	 variously	
used	 as	 dyeing	 mordant,	 catalysts,	 pigments,	 fertilizer,	 feeds,	
disinfectants,	 in	 tanning,	 soil	 conditioning,	 and	 treatment	 of	
sewage	and	industrial	wastes	[8].	Divalent	iron	is	a	cofactor	in	
heme	enzymes	such	as	catalyses	and	cytochrome	C,	and	in	non‐
heme	enzymes	such	as	aldolase	and	tryptophan	oxygenase	[7].	
In	 human	 iron	 is	 an	 essential	 component	 involved	 in	 oxygen	
transport	 [9,10].	 It	 is	 also	 essential	 for	 the	 regulation	 of	 cell	
growth	 and	 differentiation	 of	 iron	 limits	 oxygen	 delivery	 to	
cells	 [11],	 resulting	 in	 fatigue,	 poor	 work	 performance	 and	
decreased	immunity	[9].	On	the	other	hand,	excess	amounts	of	
iron	 can	 result	 in	 toxicity	 and	 even	 death	 [12].	 Toxicology	

considerations	 are	 important	 in	 terms	 of	 iron	 deficiency	
(anemia)	 and	 accidental	 acute	 exposure	 and	 chronic	 iron	
overload	 due	 to	 idiopathic	 hemochromatosis	 or	 as	 a	
consequence	 of	 excess	 dietary	 iron	 or	 frequent	 blood	
transfusions.	The	immediate	cause	of	death	from	the	inorganic	
compounds	 of	 iron	 in	 animals	 is	 respiratory	 failure.	 Clinical	
signs	 preceding	 death	 are	 anorexia	 oligodipsia,	 oliguria,	
alkalosis,	 diarrhea,	 loss	 of	 body	 weight,	 hypothermia	 and	
alternating	 irritability	 and	 depression.	 In	 human	 poisonings,	
symptoms	 of	 iron	 intoxication	 include	 vomiting,	 cirrhosis	 of	
liver,	 hemochromatosis,	 diarrhea,	 lethargy,	 coma,	 irritability,	
seizures	and	abdominal	pain	[8].	All	these	findings	cause	great	
concern	 regarding	 public	 health,	 demanding	 accurate	
determination	of	this	metal	ion	at	trace	and	ultra‐trace	levels.	

Spectrophotometry	 is	 one	 of	 the	 most	 powerful	 tools	 in	
chemical	 analysis.	 1,2‐dihydroxy‐benzene‐3,5‐disulfonic	 acid	
(Tiron)	 (Scheme	 1)	 has	 not	 previously	 been	 used	 for	 the	
spectrophotometric	 determination	 of	 iron.	 The	 method	
possesses	 distinct	 advantages	 over	 existing	 methods	 [13‐18]	
with	 respect	 to	 sensitivity,	 selectivity,	 range	of	determination,	
simplicity,	speed,	pH/acidity	range,	thermal	stability,	accuracy,	
precision,	 and	 ease	 of	 operation.	 A	 comparison	 between	
existing	methods	[13‐18]	and	the	present	method	 is	shown	in	
Table	1.		



102	 Zannat	and	Ahmed	/	European	Journal	of	Chemistry	5	(1)	(2014)	101‐110	
	

	
Table	1.	Summary	of	the	existing	spectrophotometric	methods	for	the	determination	of	iron.	
Reagent	 max	

(nm)	
Ɛ	
(L/mol.cm)	

Beer’s	law	
(mg/L)	

Interference	 Remarks	 Reference	

1‐Nitroso‐2‐naphtol	 420	 2.57104	 0.05‐3.5	 Many	 i)	pH	dependent		
ii)	Less	sensitive	
iii)Less	selective	due	to	
much	interference	

[13]	

2,2‐Dipyridine	 522	 8.7103	 0.1‐50 Many i)	pH‐ dependent	
ii)	Less	selective	due	to	
much	interference		
iii)	Less	sensitive		

[14]

Dimethyldithi‐ocarbamate	 426	 1.6103	 0.5‐20	 Many	 i)	Solvent	extractive	
ii)	Less	selective	due	to	
much	interference	
iii)	Lengthy	and	time	
consuming		

[15]	

Ferrozine		 560	 4104	 0‐10 Co(II),	Zn,	Mn,	Cd,	
Pb	etc.	

i)	Less	selective	due	to	
much	interference	
ii)	Solvent	extractive	
iii)	Less	sensitive	

[16]

5‐Nitro‐6‐amino‐1,	10‐phenanthrolone	
(NAP)	

520	 1.39103	 1‐4 Cu	(II)	
Ni	(II)		
Co	(II),	Zn,		
Mn,	Al,	Ca,	Mg		

i)Temperature	dependent		
ii)	pH	dependent		
ii)	Less	selective	due	to	
much	interference		
iv)Less	sensitive	

[17]

1,2‐Dihadroxy‐3,4	diketocyclobutence	
(squaric	acid)	

515	 3.95103	 0.5‐20	 Many	 i)	pH	dependent		
ii)	Less	sensitive		
iii)	Solvent	extractive	
iv)	Less	selective	due	to	
much	interference		
v)Lengthy	and	time	
consuming		

[18]	

Tiron	[1,2‐Dihydroxybenzene‐3,5‐
disulfonic	acid(disodium	salt	hydrate)	
(Present	method)	

665	 6105	 0.02‐70	
	

Using	suitable	
masking	agents,	
the	reaction	can	be	
made	highly	selective	

i)	Non‐extractive	and	very	
simple	
ii)Highly	sensitive	
iii)Highly	selective	
iv)	Aqueous	reaction	
media	
v)	Simple	and	rapid	
method		

Present	
Method		

	
	

	
	

1,2‐Dihydroxybenzene‐3,5‐disulfonic	acid	(Disodium	salt	hydrate)	(Tiron)	

Scheme	1	
	
	

The	method	is	based	on	the	reaction	of	non‐absorbent	tiron	
in	a	slightly	acidic	solution	(2×10‐6	‐	2×10‐5	mol/L	H2SO4)	with	
iron(III)	 to	 produce	 a	 highly	 absorbent	 navy‐blue	 chelate	
product	followed	by	a	direct	measurement	of	the	absorbent	in	
an	aqueous	solution.	With	suitable	masking,	the	reaction	can	be	
made	 highly	 selective	 and	 the	 reagent	 blank	 solutions	 do	 not	
show	any	absorbance.	
	
2.	Experimental	
	
2.1.	Apparatus	
	

A	 Shimadzu	 (Kyoto,	 Japan)	 (Model	 ‐	 1800)	 double	 beam	
UV/VIS	 the	 recording	 spectrophotometer	 and	 a	 Jenway	
(England	UK)	(Model	‐	3010)	pH‐meter	with	a	combination	of	
electrodes	were	used	for	the	measurements	of	absorbance	and	
pH,	 respectively.	 A	 Shimadzu	 (Model:	 AA7000)	 atomic	
absorption	 spectrometer	 equipped	 with	 a	 microcomputer‐
controlled	air‐acetylene	flame	was	used	 for	comparison	of	 the	
results.	
	
2.2.	Reagents	and	solutions	
	

All	chemicals	used	were	of	analytical‐reagent	grade	or	 the	
highest	 purity	 available.	Doubly	distilled	deionized	water	was	
used	 throughout	 this	 study.	 Glass	 vessels	 were	 cleaned	 by	
soaking	in	acidified	solutions	of	KMnO4	or	K2	Cr2O7,	followed	by	
washing	with	concentrated	HNO3,	and	was	rinsed	several	times	
with	 high‐purity	 deionized	 water.	 Stock	 solutions	 and	
environmental	 water	 samples	 (1000	 mL	 each)	 were	 kept	 in	
polypropylene	 bottles	 containing	 1	mL	 of	 concentrated	HNO3.	
More	 rigorous	 contamination	 control	 was	 applied	 when	 the	
iron	levels	in	specimens	were	low.	
	
2.2.1.	Tiron	solution		
	

Tiron	 solution	 (3×10‐3	mol/L)	was	prepared	by	dissolving	
the	requisite	amount	of	tiron	(BDH	Chemicals,	purity	>	99%)	in	
a	 known	 volume	 of	 doubly	 distilled	 deionized	 water.	 More	
dilute	 solutions	 of	 the	 reagent	 were	 prepared	 as	 and	 when	
required.	
	
2.2.2.	Iron	(II)	standard	solution		
	

A	100	mL	amount	of	stock	solution	(1	mg/mL)	of	divalent	
iron	 was	 prepared	 by	 dissolving	 497	 mg	 of	 purified‐grade	



Zannat	and	Ahmed	/	European	Journal	of	Chemistry	5	(1)	(2014)	101‐110	 103	
 

(Merck	 pro	 analysis	 grade)	 FeSO4.7H2O	 in	 deionized	 water.	
More	dilute	 standard	solutions	were	prepared	by	appropriate	
dilution	 of	 aliquots	 from	 the	 stock	 solution	 with	 deionized	
water	as	and	when	required.	Concentrations	(1.79×10‐2	mol/L)	
were	 checked	 using	 the	 standard	 potassium	 dichromate	
solution	[19].	
	
2.2.3.	Iron	(III)	standard	solution	
	

A	100	mL	amount	of	stock	solution	(1	mg/mL)	of	trivalent	
iron	 was	 prepared	 by	 dissolving	 490	 mg	 of	 ferric	 chloride	
(FeCl3.6H2O)	(Aldrich	A.C.S.	grade)	in	doubly	distilled	deionized	
water.	 Aliquots	 of	 this	 solution	 were	 standardized	 with	
potassium	 dichromate	 solution	 [19].	 More	 dilute	 standard	
solutions	 (1.79×10‐2	 mol/L)	 were	 prepared	 from	 this	 stock	
solution	as	and	when	required.	
	
2.2.4.	Potassium	permanganate	solution	
	

A	 1%	 potassium	 permanganate	 (Merck)	 solution	 was	
prepared	 by	 dissolving	 in	 deionized	 water.	 Aliquots	 of	 this	
solution	were	standardized	with	oxalic	acid.	
	
2.2.5.	Potassium	dichromate	solution	
	

A	100	mL	amount	of	stock	solution	(0.1	N)	was	prepared	by	
dissolving	500	mg	of	 finely	powdered	K2Cr2O7	 (Merck)	 in	100	
mL	deionized	water.	
	
2.2.6.	Sodium	azide	solution	
	

Sodium	azide	solution	(2.5%	w:v)	(Fluka	purity	>	99%)	was	
freshly	 prepared	 by	 dissolving	 2.5	 g	 in	 100	 mL	 of	 deionized	
water.	
	
2.2.7.	Tartrate	solution	
	

A	 100	 mL	 stock	 solution	 of	 tartrate	 (0.01%	 w:v)	 was	
prepared	 by	 dissolving	 10	 mg	 of	 potassium	 sodium	 tartrate	
tetra	hydrate	(ACS‐grade,	99%)	in	100	mL	deionized	water.	
	
2.2.8.	Aqueous	ammonium	solution	
	

A	 100	mL	 solution	 of	 aqueous	 ammonia	was	prepared	 by	
diluting	 10	 mL	 concentrated	 NH4OH	 (28‐30%,	 ACS	 grade)	 to	
100	 mL	 with	 deionized	 water.	 The	 solution	 was	 stored	 in	 a	
polypropylene	bottle.	
	
2.2.9.	EDTA	solution	
	

A	 100	 mL	 stock	 solution	 of	 EDTA	 (0.01%	 w:v)	 was	
prepared	by	dissolving	10	mg	ethylenediaminetetraacetic	acid	
(A.C.S.‐grade,	 	 99%)	 as	 disodium	 salt	 dihydrate	 in	 100	 mL	
deionized	water.	
	
2.2.10.	Other	solutions	
	

Solutions	 of	 a	 large	 number	 of	 inorganic	 ions	 and	
complexing	 agents	were	 prepared	 from	 their	 analytical	 grade	
or	 equivalent	 grade	 water	 soluble	 salts	 (or	 the	 oxides	 and	
carbonates	 in	 hydrochloric	 acid);	 those	 of	 niobium,	 tantalum,	
titanium,	zirconium	and	hafnium	were	specially	prepared	from	
their	 corresponding	 oxides	 (Specupure,	 Johnson	 Matthey)	
according	to	the	recommended	procedures	of	Mukharji	[20].	In	
the	 case	 of	 insoluble	 substances,	 special	 dissolution	methods	
were	adopted	[21].	
	
2.3.	Procedure	
	

A	 volume	 of	 0.1‐1.0	 mL	 of	 a	 neutral	 aqueous	 solution	
containing	 0.2‐700	 μg	 of	 iron(III)	 in	 a	 10mL	 calibrated	 flask	

was	 mixed	 with	 a	 1:75‐1:350	 fold	 molar	 excess	 of	 the	 tiron	
reagent	solution	(preferably	2	mL	of	3×10‐3	mol/L)	followed	by	
the	 addition	 of	 0.2‐2.0	mL	 (preferably	 1	mL)	 of	 1×10‐4	mol/L	
sulfuric	 acid.	After	one	minute	 the	mixture	was	diluted	 to	 the	
mark	with	deionized	water.	The	absorbance	was	measured	at	
665	 nm	 against	 a	 corresponding	 reagent	 blank.	 The	 iron	
content	 in	 an	 unknown	 sample	 was	 determined	 using	 a	
concurrently	prepared	calibration	graph.	
	
2.4.	Sample	collection	and	preservation	
	

Water:	Water	 samples	were	 collected	 in	polythene	bottles	
from	 shallow	 tube‐wells,	 tap‐wells,	 river,	 sea	 and	 drain	 of	
different	places	of	Bangladesh.	After	collection,	HNO3	(1	mol/L)	
was	added	as	preservative.	

Blood	and	urine:	Blood	and	urine	samples	were	collected	in	
polypropylene	 bottles	 from	 effected	 persons	 of	 Chittagong	
Medical	 College	 Hospital,	 Bangladesh.	 Immediately	 after	
collection	 they	were	 stored	 in	a	 salt‐ice	mixture	and	 latter,	 at	
the	laboratory,	were	kept	at	‐20	°C.	

Soil:	 Soil	 (surface)	 samples	 were	 collected	 from	 different	
locations	 in	 Bangladesh.	 Samples	 were	 dried	 in	 air	 and	
homogenized	with	a	mortar.	

Food:	 Food	 samples	 were	 collected	 from	 local	 market	 of	
Chittagong	in	Bangladesh.	
	
3.	Results	and	discussions	
	
3.1.	Absorption	spectra	
	

The	absorption	spectra	of	the	Fe(III)‐tiron	system	in	1×10‐4	
mol/L	 H2SO4	 medium	 were	 recorded	 using	 the	
spectrophotometer.	The	absorption	spectra	of	the	Fe(III)‐tiron	
is	a	symmetric	curve	with	maximum	absorbance	at	665	nm	and	
the	 average	 molar	 absorption	 coefficient	 of	 6×105	 L/mol.cm	
was	shown	in	Figure	1.	The	reagent	blank	exhibited	negligible	
absorbance	despite	having	wave	length	in	the	same	region.	The	
reaction	 mechanism	 of	 the	 present	 method	 is	 as	 reported	
earlier	[22].	
	

	
	
Figure	 1.	 A	 and	 B	 Absorbance	 spectra	 of	 Fe(III)‐Tiron	 system	 and	 the	
reagent	blank	(λmax	=	665	nm)	in	aqueous	solutions,	respectively.	
	
	
3.2.	Effect	of	acidity	
	

Of	 the	 various	 acids	 (nitric,	 hydrochloric,	 sulfuric	 and	
phosphoric)	studied.	Sulfuric	acid	was	found	to	be	the	best	acid	
for	 the	 system.	 The	 absorbance	 was	 maximum	 and	 constant	
when	the	10	mL	of	solution	(1	mg/L)	contained	0.2‐2.0	mL	of	
1×10‐4	 M	 H2SO4	 at	 room	 temperature.	 Outside	 this	 range	 of	
acidity,	 the	 absorbance	 decreased	 (Figure	 2).	 For	 all	
subsequent	measurements,	1	mL	of	1×10‐4	M	H2SO4	was	added.	



104	 Zannat	and	Ahmed	/	European	Journal	of	Chemistry	5	(1)	(2014)	101‐110	
	

	
Table	2.	Selected	analytical	parameters	obtained	with	the	optimization	experiments.	
Parameters	 Studied	range	 Selected	value	
Wavelength,	λmax	(nm)	 200‐800 665
Acidity	(M	H2SO4)	 110‐7‐310‐5 210‐6‐210‐5 (preferably,	110‐4)	
pH	 4.58	‐ 3.76 4.5	‐ 5.5	(preferably,	5.00)	
Time	(h)	 0	‐ 72 1	min	‐ 24	h	(preferably,	1	min)	
Temperature	(°C)	 10‐90 25±5
Reagent	(fold	molar	excess,	M:R)	 1:1‐1:350 1:75	‐ 1:350	(preferably,	1:75)	
Molar	absorptivity	(L/mol.cm)	 5.25105‐6.75105 6105	
Linear	range	(mg/L)	 0.001‐100	 0.02	‐	70	
Detection	limit	(µg/L)	 0.01	‐	100	 1.0	
Sandell’s	sensitivity	(ng/cm2)	 1	‐ 100 10		
Reproducibility	(%	RSD)	 0	‐ 5 0	‐ 3
Regression	co‐efficient	(R2)	 0.9996‐0.9998 0.9997
	
	
	

	
	

Figure	2.	Effect	of	acidity	on	the	absorbance	of	Fe(III)‐Tiron	system	(0.0001	
M	H2SO4).	
	
	
3.3.	Effect	of	time	
	

The	reaction	was	very	fast.	Constant	maximum	absorbance	
was	obtained	within	 few	seconds	 after	 the	dilution	 to	volume	
and	 remained	 strictly	 unaltered	 for	 over	 24	 hours.	 A	 longer	
period	of	time	was	not	studied.	
	
3.4.	Effect	of	temperature	
	

Effect	 of	 various	 temperatures	 (10‐90	 °C)	 on	 Fe(III)‐tiron	
system	 was	 studied.	 The	 iron(III)‐tiron	 system	 attained	
maximum	 and	 constant	 absorbance	 at	 room	 temperature	
(25±5	°C).	
	
3.5.	Effect	of	reagent	concentration	
	

Different	 molar	 excesses	 of	 tiron	 were	 added	 to	 a	 fixed	
metal	 ion	 concentration	 and	 absorbances	 were	 measured	
according	to	the	standard	procedure.	It	was	observed	that	at	1	
mg/L	 Fe(III)	 metal,	 the	 reagent	 molar	 ratios	 of	 1:75‐1:350	
produced	 a	 constant	 absorbance	 of	 the	 Fe‐chelate	 (Figure	 3).	
For	 all	 subsequent	 measurements,	 2	 mL	 of	 3×10‐3	 M	 tiron	
reagent	was	added.	
	
3.6.	Effect	of	metal	concentration	(Beer’s	law)	
	

The	well‐known	 equation	 for	 spectrophotometric	 analysis	
in	 very	dilute	 solutions	derived	 from	Beer’s	 law.	The	effect	of	
metal	 concentration	 was	 studied	 over	 0.01‐100	 mg/L	
distributed	 in	 four	different	sets	(0.01‐	0.10,	0.1‐1.0,	1‐10	and	
10‐100	 mg/L)	 for	 convenience	 of	 measurement.	 The	
absorbance	 was	 linear	 for	 0.02‐70.00	 mg/L	 of	 Fe(III)	 at	 665	
nm.	The	molar	 absorption	 coefficient	 and	 Sandell’s	 sensitivity	
[23]	were	6×105	L/mol.cm	and	10	ng/cm2,	respectively.	Of	the	
four	 calibration	curves,	 the	 first	 three	pass	 through	 the	origin	

and	 the	 fourth	 (Figure	 4)	 one	 shows	 the	 deviation	 from	
linearity.	The	selected	analytical	parameters	obtained	with	the	
optimization	experiments	are	summarized	in	Table	2.	
	

	
	
Figure	3.	Effect	of	 reagent	 (Fe(III):Tiron	molar	 concentration	 ratio)	on	 the	
absorbance	of	Fe(III)‐Tiron	system.	
	

	
	

Figure	4.	Calibration	graph‐D,	10‐70	mg/L	of	iron(III).
	
	
3.7.	Effect	of	foreign	ions	
	

The	effect	of	over	50	anions,	cations,	and	complexing	agents	
on	the	determination	of	only	1	mg/L	of	Fe(III)	was	studied.	The	
criterion	for	interference	[24]	was	an	absorbance	value	varying	
by	more	than	5%	from	the	expected	value	for	Fe(III)	alone.	The	
results	 are	 summarized	 in	 Table	 3.	 As	 can	 be	 seen,	 a	 large	
number	of	ions	have	no	significant	effect	on	the	determination	
of	 iron.	Only	 iron(II)	 interferes	 and	 in	 order	 to	 eliminate	 this	
interference	 1,10‐phenanthroline	 is	 used	 as	 masking	 agents.	
During	the	 interference	studies,	 if	a	precipitate	was	 formed,	 it	
was	removed	by	centrifugation.		



Zannat	and	Ahmed	/	European	Journal	of	Chemistry	5	(1)	(2014)	101‐110	 105	
 

	
Table	3.	Tolerance	limits	a	of	foreign	ions,	tolerance	ratio[Species	(x)/Fe	(w:w)].	
Species,	x	 Tolerance	ratio,	[Species	(x)/Fe	(w:w)] Species,	x Tolerance	ratio,	[Species	(x)/Fe	(w:w)]
Acetate	 100	 Lead(II)	 100
Aluminum	 100	 Magnesium		 100	
Ammonium		 1000	 Manganese(II)	 1000	
Arsenic(III)	 50	 Manganese(VII)	 100	
Arsenic	(V)	 500	 Mercury(II)	 100	
Ascorbic	acid	 500	 Molybdenum(VI) 50	
Antimony	 50	 Nickel(II) 500
Azide	 100	 Nitrate 1000
Barium	 100	 Oxalate	 20
Beryllium(II)	 100	 Phosphate 1000
Bromide	 1000	 Potassium 1000
Phosphate	 100	 Selenium(IV) 25
Cadmium	 100	 Selenium(VI)	 1000	
Calcium	 200	 Silver 100
Cesium		 1000	 Sodium	 50	
Chloride	 1000	 Strontium	 100	
Chromium(III)	 100	 Tartrate 100
Chromium(VI)	 100	 Tellurium 500
Citric	acid	 50	 Thiocyanate 10
Copper	(II)	 100	 Tin(II)	 50
Cyanide	 1000	 Tin(IV) 100
Carbonate	 50	 Titanium(IIII) 100
Cerium(III)	 100	 Tungsten(VI) 100
EDTA	 10	 Vanadium(V)	 50	
Fluoride	 1000	 Zinc	 100	
Iron(II)	 50	b	 Lithium	 100	
Iodide	 1000	 Nitrite	 1000	
a	Tolerance	limit	was	defined	as	ration	that	causes	less	than	±5	percent	interference.	
b	With	1,10‐phenanthroline.	
	
	
Table	4.	Determination	of	iron	in	some	synthetic	mixtures.	
Sample	 Composition	of	mixtures(mg/L)	 Iron(III)	(mg/L)	

Added Found	a Recovery	±	s	b	(%)	
A	 Fe3+	 0.50

1.00	
0.49
1.00	

98±1.0	
100±0.0	

B	 As	in	A	+	Cu2+	(50)	+	Ca	(50)	 0.50
1.00	

0.50
0.99	

100±0.0	
99±1.0	

C	 As	in	B	+	Mn2+	(25)	+	Ni2+	(25)	 0.50
1.00	

0.49
1.02	

98±1.0	
102±1.0	

D	 As	in	C	+	K	(25)	+	Co2+	(25)	+	Hg2+	(25)	 0.50	
1.00	

0.52	
1.03	

104±1.3	
103±1.0	

E	 As	in	D	+	Zn	(25)	+	Pb2+	(25)	+	Cd	(25)		 0.50	
1.00	

0.54	
1.08	

108±1.0	
108±1.2	

F	 As	in	E	+	Mg	(25)	+	Al	(25)	+	Sr	(25)	 0.50
1.00	

0.55
1.10	

110±1.8	
110±1.5	

a	Average	of	five	analysis	of	each	sample.	
b	The	measure	of	precision	is	the	standard	deviation	(s).	
	

	
The	 amount	 mentioned	 is	 not	 the	 tolerance	 limit	 but	 the	

actual	 amount	 studied.	 However,	 for	 those	 ions	 whose	
tolerance	 limit	 has	 been	 studied,	 their	 tolerance	 ratios	 are	
given	in	Table	3.	

	
3.8.	Composition	of	the	absorbent	complex	
	

Job’s	 method	 [25]	 of	 continuous	 variation	 and	 the	 molar	
ratio	[26]	method	were	applied	to	ascertain	the	stoichiometric	
composition	of	the	complex	(Figure	5).	A	Fe‐tiron	complex	was	
indicated	by	both	methods.	
	
3.9.	Precision	and	accuracy	
	

The	 precision	 of	 the	 present	 method	 was	 evaluated	 by	
determining	different	concentrations	of	 iron	(each	analyzed	at	
least	five	times).	The	relative	standard	deviation	(n	=	5)	was	0‐
3%	 for	 0.2‐700.0	 μg	 of	 iron(III)	 in	 10	mL,	 indicating	 that	 this	
method	is	highly	precise	and	reproducible.		

The	 detection	 limit	 (3s/S	 of	 the	 blank)	 and	 Sandell’s	
sensitivity	 (concentration	 for	 0.001	 absorbance	 unit)	 for	
iron(III)	were	found	to	be	1	µg/L	and	10	ng/cm2,	respectively.	
The	method	was	tested	by	analyzing	several	synthetic	mixtures	
containing	 iron(III)	and	diverse	ions	(Table	4).	The	results	 for	
total	iron	were	in	good	agreement	with	certified	values	(Table	
5).	The	reliability	of	our	Fe‐chelate	procedure	was	also	 tested	

by	recovery	studies.	The	average	percentage	recovery	obtained	
for	 addition	 of	 iron(III)	 spike	 to	 some	 environmental	 water	
samples	was	quantitative	as	shown	in	(Table	6)	The	results	of	
biological	analyses	by	the	spectrophotometric	method	were	in	
excellent	agreement	with	those	obtained	by	AAS	(Table	7).		
	

	
	

	Figure	5.	Composition	of	Fe(III)‐Tiron	complex	by	the	Job’s	method.
	
	



106	 Zannat	and	Ahmed	/	European	Journal	of	Chemistry	5	(1)	(2014)	101‐110	
	
	
Table	5.	Determination	of	iron	in	certified	reference	materials.	
Sample	no	 Certified	reference	materials	(Composition,	%) Iron	(%)	 RSD

(%)	Certified	value	 Found	a
1	 BAS‐CRM‐20	b;	High	speed	Steel	

(Al=90.5,	Fe=0.43,	Cu=4.1,	Ni=1.9,	Mn=0.19,	Si=0.24	
0.43 0.45	 2.5

2	 YSBC	19716	c	High	tensile	steel		
(Fe=34.26,	Zn=36.24,	Si=0.38,	Cd=1.2,	Sb=48.57,	S=0.95	and	F=0.32)		

1.56 1.52	 1.5

3	 YSBC	19716	c	High	tensile	steel		
(Fe=34.26,	Zn=36.24,	Si=0.38,	Cd=1.2,	Sb=48.57,	S=0.95	and	F=0.32)	

34.46	 34.12	 1.0	

4	 BY	0110‐1	c	High	tensile	steel		
(Zn=42.98,	Si=19.89,	Fe=4.13,	Pb=0.351,	Sn=0.06,	Cd=0.04,	As=0.024,	Sn=0.06	and	Cu=14)		

4.13	 4.06	 1.4	

5	 GSBD	33001‐94	c	High	tensile	steel		
(Fe=9.53,	Si=14.64,	Al=9.29,	Ca=1.04,	Mg=21.49	and	Cr=32.79)		

9.53 9.38	 1.2

a	Average	of	five	replicate	determinations.
	

b	The	measure	of	precision	is	the	relative	standard	deviation	(RSD).	
c	These	CRMs	were	obtained	from	Beijing	NCS	Analytical	Instruments	Co.	Ltd,	China.	
	
	
Table	6.	Determination	of	iron	in	some	environmental	water	samples.	
Sample	 Iron	(µg/L)	 Recovery	±	s	(%)	 sr	b	(%)	

Added Found	a
Tap	water	 0	

100	
500	

145.0	
248.0	
650.0	

1010.7	
1000.0	

0.41	
0.00	

Rain	water	 0
100	
500	

5.0
108.0	
510.0	

1030.8	
1010.5	

0.22	
0.29	

Well	water	 0
100	
500	

10.0
110.0	
512.0	

1000.0	
100.40.9	

0.00	
0.21	

River	water	 Karnaphuly	
(upper)	

0	
100	
500

55.0	
160.0	
555.0

1030.9	
99.90.5	

0.23	
0.29	

Karnaphuly	
(lower)	

0
100	
500	

60.0
160.0	
568.0	

1000.0	
1010.5	

0.00	
0.20	

Halda	(upper)	 0
100	
500	

40.0
140.0	
550.0	

1000.0	
1020.8	

0.00	
0.18	

Halda(lower)	 0	
100	
500

45.0	
148.0	
545.0

1020.6	
1000.0	

0.21	
0.00	

Sea	water	 Bay	of	Bengal	
(upper)	

0	
100	
500	

10.0	
110.0	
520.0	

1000.0	
1020.8	

0.00	
0.31	

Bay	of	Bengal	
(lower)	

0
100	
500	

12.0
112.0	
520.0	

1000.0	
1010.5	

0.00	
0.16	

Drain	water	 KSRM	c	 0
100	
500

560.0
670.0	
1055.0

1010.8	
990.5	

0.35	
0.45	

Eastern	Refinery	d	 0
100	
500	

160.0
260.0	
570.0	

1000.0	
1020.5	

0.00	
0.31	

KDS	Texile	e	 0	
100	
500	

135.0	
235.0	
640.0	

1000.0	
1020.7	

0.00	
0.28	

TSP	Complex	f	 0	
100	
500

395.0	
500.0	
895.0

1010.7	
1000.0	

0.15	
0.00	

a	Average	of	five	replicate	determinations.	
b	The	measure	of	precision	is	the	relative	standard	deviation	(sr).	
c	Kabir	Steel	Re‐Rolling	Mills,	Chittagong.	
d	Estern	Refinary	Ltd.,	Patenga,	Chittagong.	
e	KDS	Textile	Ltd.,	Oxygen,	Chittagong.	
f		TSP	complex	Ltd.,	Patenga,	Chittagong.	
	
	
3.10.	Applications	
	

The	 proposed	 method	 was	 successfully	 applied	 to	 the	
determination	 of	 iron(III)	 in	 a	 series	 of	 synthetic	mixtures	 of	
various	 compositions	 (Table	 4)	 and	 also	 in	 a	 number	 of	 real	
samples	 e.g.	 several	 Certified	 Reference	 Materials	 (CRMs)	
(Table	5).	The	method	was	also	extended	to	the	determination	
of	 iron	 in	 a	 number	 of	 environmental,	 biological,	 pharma‐
ceutical,	 soil	 and	 food	 samples.	 In	 view	 of	 the	 unknown	
composition	 of	 environmental	 water	 samples,	 the	 same	
equivalent	 portions	 of	 each	 such	 samples	 were	 analyzed	 for	
iron	content;	the	recoveries	in	both	the	“spiked”	(added	to	the	
samples	 before	 the	 mineralization	 or	 dissolution)	 and	 the	

“unspiked”	 samples	 are	 in	 good	 agreement	 (Table	 6).	 The	
results	 of	 biological	 analyses	 by	 spectrophotometric	 method	
were	found	to	be	in	excellent	agreement	with	those	obtained	by	
AAS	 (Table	 7).	 The	 results	 of	 soil	 sample	 analyzed	 by	 the	
spectrophotometric	method	are	shown	in	Table	8.	The	results	
of	food	and	pharmaceutical	samples	by	the	spectrophotometric	
method	are	shown	in	Table	9	and	10.	The	results	of	speciation	
of	iron(II)	and	iron(III)	in	mixtures	are	shown	in	Table	11.	The	
results	of	 speciation	of	 iron(II)	and	 iron(III)	 in	mixtures	were	
highly	 reproducible	 (Table	 11).	 Hence,	 the	 precision	 and	
accuracy	of	the	method	were	excellent	
	

	



Zannat	and	Ahmed	/	European	Journal	of	Chemistry	5	(1)	(2014)	101‐110	 107	
 

	
Table	7.	Concentration	of	iron	in	blood	and	urine	samples.	
Serial	no	 Sample	 Iron	(mg/L)	 Sample	source	a	

AAS	(n	=	5)	 Proposed	method	(n	=	5)
Found	 RSD,	%	 Found	 RSD	b,	%	

1	 Blood	
Urine	

2.0	
0.51	

1.0	
1.2	

2.10
0.55	

1.0
1.3	

Kidney	diseases	patient	(Female)	

2	 Blood	
Urine	

0.65	
0.17	

1.5	
1.7	

0.70	
0.18	

1.5	
1.8	

Anemia	patient	(Female)	

3	 Blood	
Urine	

1.0	
0.25	

1.3	
1.5	

1.05	
0.27	

1.3	
1.6	

Pregnant	women	

4	 Blood	
Urine	

4.95	
1.25	

1.0	
1.5	

4.85
1.21	

1.2
1.8	

Liver	cirrhosis	patient	(Male)	

5	 Blood	
Urine	

1.25	
0.30	

1.0	
1.8	

1.29
0.33	

1.2
1.5	

Normal (Male)	

a	The	samples	were	from	Chittagong	Medical	College	Hospital,	Chittagong.	
b	The	measure	of	precision	is	the	relative	standard	deviation(RSD).	
	

	
Table	8.	Determination	of	iron	in	some	surface	soil	samples.	
Serial	no	 Iron	(mg/kg)	a	 Sample	source	c	
S1	 21.01.5	b	 Marine	soil

(Bay	of	Bengal,	Chittagong,	Bangladesh)	
S2	 41.51.2	 Traffic	soil

(Bahaddarhat,	Bus	Terminal,	Chittgong)	
S3	 34.51.4	 Estuarine	soil

(Junction	of	Bay	of	Bengal	and	River	Karnafuly,	Chittagong,	Bangladesh)	
S4	 38.51.0	 Agricultural	soil	

(Chittagong	University	Campus)	
S5	 78.82.0	 Industrial	soil	

(Bangladesh	Steel	Re‐rolling	Mills	Ltd.,	Chittagong,	Bangladesh)	
a	Average	of	five	analysis	of	each	sample.	
b	The	measure	of	precision	is	the	standard	deviation	(±s).	
c	Composition	of	soil	samples:	C,	N,	P,	K,	Na,	Ca,	Mg,	Fe,	Pb,	Cu,	Zn,	Mn,	Mo,	Co,NO3,	NO2,	SO4,	etc.		
	

	
Table	9.	Determination	of	iron	in	some	food	samples.	
Serial	no	 Sample	 Iron	(mg/kg	or	mg/L) Sample	source	

Found	a	s	
1	 Cow’s	milk	b	

(Boss	indicus)	
2.01.5	 Chittagong	Market	

2	 Banana	
(Musa	acuminata)	

10.02.0	 Chittagong	Market	

3	 Tomato	
(Licopersicon	esculentum)	

18.01.0	 Chittagong	Market	

4	 Date	juice	b	
(Phoenix	dactylifera)	

9.01.8	 Chittagong	Market	

5	 Arum	
(Arum	discorides)		

14.01.4	
	

Chittagong	Market	

6	 Guava	
(Psidium	guajava)	

12.01.3	 Chittagong	Market	

7	 Egg(Hen)	
(Gallious	domesticus)	

0.061.6	
	

Chittagong	Market	

a	Average	of	the	five	replicate	determinations	of	each	sample.	
b	Values	in	mg/L.	
	

	
Table	10.	Determination	of	iron	in	some	pharmaceutical	samples.	
Serial	no	 Compositionof	Tablet	 Trade	name Iron	(µg/g	or	µg/mL)	 RSD,	%

Reported	 Found	
1	 Ferrous	sulphate	(150	mg	per	0.45	g)	a	 Zif

(Square	Pharmaceuticals	Ltd.)	
33.34 338.0	 2.0

2	 Iron(III)hydroxide	(10	mg/mL)	b	 Aritone	ZI	
(Incepta	Pharmaceuticals	Ltd.)	

10,000	 10,050	 1.5	

3	 Carbonyl	Iron	(51	mg	per	0.45	g)	a	 Glory	
(Orion	Pharma	Ltd.)	

113.34	 115.0	 2.5	

a	Values	in	µg/g.	
b	Values	in	µg/mL.	
	

	
	3.10.1.	Determination	of	iron	in	synthetic	mixtures	
	

Several	 synthetic	 mixtures	 of	 varying	 compositions	
containing	iron	and	diverse	ions	of	known	concentrations	were	
determined	by	the	present	method	and	the	results	were	found	
to	 be	 highly	 reproducible.	 The	 results	 are	 shown	 in	 Table	 4.	
Accurate	recoveries	were	achieved	in	all	solutions.	
	
3.10.2.	Determination	of	iron	in	brass,	alloys	and	steels	
(Certified	reference	materials)	
	

A	 0.1	 g	 amount	 of	 a	 brass	 or	 alloy	 or	 steel	 sample	
containing	 0.43‐34.26%	 of	 iron	 was	 weighed	 accurately	 and	

placed	 in	 a	 50	 mL	 erlenmeyer	 flask	 following	 a	 method	
recommended	 by	 Parker	 [27].	 To	 it,	 10	 mL	 of	 concentrated	
HNO3,	 1	mL	 of	 concentrated	H2SO4	 and	1‐2	mL	of	 1%	KMnO4	
were	 added	 to	 oxidize	 Fe(II)	 to	 Fe(III),	 excess	 of	 KMnO4	was	
removed	 by	 addition	 of	 1‐2	 mL	 of	 freshly	 prepared	 2.5%	
sodium	 azide	 solution	 and	 carefully	 covering	 the	 flask	with	 a	
watch	glass	until	the	brisk	reaction	subsided.	The	solution	was	
heated	 to	drive	off	excess	azide	solution	and	simmered	gently	
after	 the	 addition	 of	 5	 mL	 of	 concentrated	 HNO3	 until	 all	
carbides	 were	 decomposed.	 The	 solution	 was	 carefully	
evaporated	 to	 dense	 white	 fumes	 to	 drive	 off	 the	 oxides	 of	
nitrogen	and	then	cooled	to	room	temperature	(25±5	°C).		

	



108	 Zannat	and	Ahmed	/	European	Journal	of	Chemistry	5	(1)	(2014)	101‐110	
	

	
Table	11.	Determination	of	iron(II)	and	iron(III)	in	mixtures.	
Serial	no	 Fe(III)	:	Fe(II)	 Fe,	taken	

(mg/L)	
Fe,	found	
(mg/L)	

Error	

Fe(III) Fe(II) Fe(III) Fe(II) Fe(III)	 Fe(II)
1	 1:1	 1.00 1.00 0.99 0.98 0.01	 0.02
2	 1:1	 1.00 1.00 1.00 1.00 0.00	 0.00
3	 1:1	 1.00 1.00 0.98 0.99 0.02	 0.01
Mean	error:	Fe(III)=	±0.01	;	Fe(II)=	±0.01	
Standard	deviation:	Fe(III)=	±0.005	;	Fe(II)=	±0.006	
1	 1:3	 1.00 3.00 0.98 2.98 0.02	 0.02
2	 1:3	 1.00 3.00 0.98 2.99 0.02	 0.01
3	 1:3	 1.00 3.00 0.99 2.98 0.01	 0.02
Mean	error:	Fe(III)=	±0.016	;	Fe(II)=	±0.016	
Standard	deviation:	Fe(III)=	±0.0058;	Fe(II)=	±0.006	
1	 1:5	 1.00 5.00 0.99 4.98 0.01	 0.02
2	 1:5	 1.00 5.00 1.00 4.99 0.00	 0.01
3	 1:5	 1.00 5.00 0.98 4.98 0.02	 0.02
Mean	error:	Fe(III)=	±0.01	;	Fe(II)=	±0.016	
Standard	deviation:	Fe(III)=	±0.005;	Fe(II)=	±0.006	
	
	

	
After	suitable	dilution	with	deionized	water,	the	contents	of	

the	Erlenmeyer	flask	were	warmed	to	dissolve	the	soluble	salts.	
The	 solution	 was	 then	 cooled	 and	 neutralized	 with	 a	 dilute	
NH4OH	 solution.	 The	 resulting	 solution	 was	 filtered,	 if	
necessary,	through	a	Whatman	No.	40	filter	paper	into	a	25	mL	
calibrated	 flask.	The	residue	was	washed	with	a	small	volume	
of	 hot	 (1+99)	 H2SO4,	 followed	 by	 water	 and	 the	 volume	 was	
made	up	to	the	mark	with	deionized	water.	

A	suitable	aliquot	(1‐2	mL)	of	the	above	solution	was	taken	
into	 a	 10	 mL	 calibrated	 flask	 and	 the	 iron	 content	 was	
determined	 as	 described	 under	 procedure.	 Based	 on	 five	
replicate	 analyses,	 average	 iron	 concentration	 determined	 by	
spectrophotometric	 method	was	 in	 close	 agreement	with	 the	
certified	values	(Table	5).	The	results	are	shown	in	Table	5.	
	
3.10.3.	Determination	of	iron	in	environmental	waters	
	

Each	filtered	(with	Whatman	No.	40)	environmental	water	
sample	(1000	mL)	evaporated	nearly	to	dryness	with	a	mixture	
of	3	mL	concentrated	H2SO4	and	10	mL	of	concentrated	HNO3	in	
a	 fume	 cupboard	 and	 1‐2	 mL	 of	 KMnO4,	 following	 a	 method	
recommended	by	Greenberg	et	al.	 [28].	 Excess	 of	 KMnO4	was	
removed	by	2.5%	 freshly	prepared	sodium	azide	solution	and	
was	heated	with	10	mL	of	deionized	water	in	order	to	remove	
excess	azide	solution	and	dissolves	the	salts.	The	solution	was	
then	 cooled	 and	 neutralized	with	 dilute	 NH4OH	 solution.	 The	
resulting	 solution	 was	 then	 filtered	 (if	 necessary)	 and	
quantitatively	 transferred	 into	 a	 25	 mL	 calibrated	 flask	 and	
made	up	to	the	mark	with	deionized	water.		

An	aliquot	(1‐2	mL)	of	this	pre‐concentrated	water	sample	
was	pipetted	into	a	10	mL	calibrated	flask	and	the	iron	content	
was	 determined	 as	 described	 under	 the	 procedure.	 The	
analyses	of	environmental	water	samples	for	iron	from	various	
sources	are	shown	in	Table	6.	

Most	spectrophotometric	method	 for	 the	determination	of	
iron	in	natural	and	sea‐water	require	pre‐concentration	of	iron	
[28].	 The	 concentration	 of	 iron	 in	 natural	 and	 sea‐water	 is	 a	
few	μg/L	 in	 Japan	 [29].	The	mean	concentration	of	 iron	found	
in	UK	drinking	water	is	 less	than	1	mg/L	(Average:	200	g/L)	
[30].	
	
3.10.4.	Determination	of	iron	in	biological	samples	
	

Human	blood	(2‐5	mL)	and	urine	(20‐30	mL)	was	collected	
in	 polyethane	 bottles	 from	 the	 affected	 persons.	 Immediately	
after	collection,	they	were	stored	in	a	salt‐ice	mixture	and	later,	
at	the	laboratory,	were	kept	at	‐20	°C.	The	samples	were	taken	
into	 a	 100	mL	micro‐Kjeldahl	 flask.	 Glass	 bead	 and	 10	mL	 of	
concentrated	nitric	 acid	were	 added	 and	 the	 flask	was	placed	
on	 the	 digester	 under	 gentle	 heating.	 When	 the	 initial	 brisk	
reaction	 was	 over,	 the	 solution	 was	 removed	 and	 cooled	

following	a	method	recommended	by	Stahr	[31].	1	mL	volume	
of	 concentrated	 sulfuric	 acid	 and	 1‐2	mL	 of	 1%	 KMnO4	were	
added	 carefully	 and	 excess	 of	 KMnO4	 was	 removed	 by	 2.5%	
freshly	 prepared	 sodium	 azide	 solution	 followed	 by	 the	
addition	of	0.5	mL	of	70%	HClO4	and	heating	was	continued	for	
at	least	½	hr	to	remove	excess	azide	solution	and	then	cooled.	
The	 solution	 of	 flask	 then	 neutralized	 with	 dilute	 NH4OH	
solution.	 The	 resultant	 solution	 was	 then	 transferred	
quantitatively	into	a	10	mL	calibrated	flask	and	made	up	to	the	
mark	with	deionized	water.	

A	 suitable	 aliquot	 (1‐2	 mL)	 of	 the	 final	 solution	 was	
pipetted	into	a	10	mL	calibrated	flask	and	the	iron	content	was	
determined	 as	 described	 under	 the	 general	 procedure.	 The	
results	 of	 biological	 analyses	 by	 the	 spectrophotometric	
method	 were	 found	 to	 be	 in	 excellent	 agreement	 with	 those	
obtained	by	AAS.	The	results	are	shown	in	Table	7.	

The	abnormally	high	value	for	the	liver	cirrhosis	patient	is	
probably	 due	 to	 the	 involvement	 of	 high	 iron	 concentration	
with	Cu	and	Zn.	Occurrence	of	such	high	iron	contents	are	also	
reported	 in	 liver	 cirrhosis	 patients	 from	 some	 developed	
countries	[7].	
	

3.10.5.	Determination	of	iron	in	soil	samples	
	

An	air	dried	homogenized	soil	sample	(100	g)	was	weighed	
accurately	 and	 placed	 in	 a	 100mL	 micro‐Kjeldahl	 flask.	 The	
sample	was	 digested	 in	 the	presence	 of	 a	 oxidizing	 agen	 (1%	
KMnO4),	 following	 the	 method	 recommended	 by	 Hesse	 [32].	
Excess	 of	 KMnO4	 was	 removed	 by	 2.5%	 freshly	 prepared	
sodium	azide	solution	and	heating	was	continued	for	at	least	½	
hr	 to	 remove	 excess	 azide	 solution	 and	 then	 cooled.	 The	
content	 of	 the	 flask	 was	 filtered	 through	 a	 Whatman	 No.	 40	
filter	paper	 into	a	25	mL	calibrated	flask	and	neutralized	with	
dilute	NH4OH	solution.	Then	the	solution	of	the	flask	was	made	
up	to	the	mark	with	deionized	water.	

Suitable	 aliquots	 (1‐2	mL)	were	 transferred	 into	 a	 10	mL	
calibrated	flask	and	a	calculated	amount	of	1×10‐4	mol/L	H2SO4	
needed	 to	 give	 a	 final	 acidity	 of	 2×10‐6	 ‐	 2×10‐5	mol/L	H2SO4	
was	added.	The	iron	content	was	then	determined	by	the	above	
procedure	 and	 quantified	 from	 a	 calibration	 graph	 prepared	
concurrently.	The	results	are	shown	in	Table	8.	
	
3.10.6.	Determination	of	iron	in	food	samples	
	

An	 air	 dried	 food	 sample	 Banana	 (50	 g),	 tomato	 (50	 g),	
aruma	(50	g),	Guava	(50	g),	egg	(1	piece)	and	Cow’s	milk	(100	
mL),	 Date	 juice	 (100	 mL)	 were	 taken	 in	 a	 100	 mL	 micro‐
Kjeldahl	 flask.	 A	 glass	 bead	 and	 10	mL	 of	 concentrated	 nitric	
acid	were	added	and	the	flask	was	placed	on	the	digester	under	
gentle	 heating.	 When	 the	 initial	 brisk	 reaction	 was	 over,	 the	
solution	 was	 removed	 and	 cooled	 following	 a	 method	



Zannat	and	Ahmed	/	European	Journal	of	Chemistry	5	(1)	(2014)	101‐110	 109	
 

recommended	 by	 Stahr	 [31].	 1	 mL	 volume	 of	 concentrated	
sulfuric	 acid	 and	 1‐2	mL	 of	 1%	 KMnO4	were	 added	 carefully	
and	 excess	 of	 KMnO4	was	 removed	 by	 2.5%	 freshly	prepared	
sodium	 azide	 solution	 followed	 by	 the	 addition	 of	 0.5	 mL	 of	
70%	 HClO4	 and	 heating	 was	 continued	 for	 at	 least	 ½	 hr	 to	
remove	 excess	 azide	 solution	 and	 then	 cooled.	 The	 resultant	
solution	 was	 then	 transferred	 quantitatively	 into	 a	 50mL	
calibrated	flask	and	made	up	to	the	mark	with	deionized	water.	

A	 suitable	 aliquot	 (1‐2	 mL)	 of	 the	 final	 solution	 was	
pipetted	into	a	10mL	calibrated	flask	and	the	iron	content	was	
determined	 as	 described	 under	 the	 procedure.	 High	 value	 of	
iron	for	tomato	is	probably	due	to	the	involvement	of	high	iron	
concentration	in	the	soil.	The	results	are	shown	in	Table	9.	
	
3.10.7.	Determination	of	iron	in	some	pharmaceutical	
samples	
	

Finished	 pharmaceutical	 samples	 (Iron	 containing	 tablet	
and	syrap)	were	quantitatively	taken	in	a	beaker.	Added	10mL	
of	conc.	nitric	acid	and	heated	to	dryness	and	then	added	10mL	
of	20%	 (v:v)	 of	 sulfuric	 acid	 and	1‐2	drops	of	 perchloric	 acid.	
The	 volume	was	 reduced	 to	 2‐5	mL	 and	 then	 cooled	 to	 room	
temperature.	 The	 solution	 was	 then	 neutralized	 with	 dilute	
NH4OH	in	the	presence	of	a	1‐2	mL	0.1%	(w:v)	KMnO4	solution	
to	oxidize	Fe(II)	 to	Fe(III)	 and	excess	of	KMnO4	was	 removed	
by	2.5%	freshly	prepared	sodium	azide	solution.	The	resulting	
solution	was	then	filtered	and	quantitatively	transferred	into	a	
25mL	volumetric	flask	and	made	up	to	the	mark	with	deionized	
water.	

A	 suitable	 aliquot	 (1‐2	 mL)	 of	 the	 final	 solution	 was	
pipetted	into	a	10mL	calibrated	flask	and	the	iron	content	was	
determined	 as	 described	 under	 the	 procedure.	 High	 value	 of	
iron	for	tomato	is	probably	due	to	the	involvement	of	high	iron	
concentration	in	the	soil.	The	results	are	shown	in	Table	10.	
	
3.10.8	Determination	of	iron	(II)	and	iron	(III)	in	mixtures	
	

Suitable	 aliquots	 (1‐2	 mL)	 of	 iron(III	 +	 II)	 mixtures	
(preferably	1:1,	1:3,	1:5)	were	taken	in	a	25	mL	conical	flask.	A	
few	drops	of	0.05	mol/L	sulfuric	acid	and	1‐3	mL	of	1%	(w:v)	
potassium	 permanganate	 solution	 were	 added	 to	 oxidize	
iron(II).	 A	 5	mL	 volume	 of	water	was	 added	 to	 the	mixtures,	
which	were	 then	heated	on	 a	 steam	bath	 for	 10‐15	min,	with	
occasional	 gentle	 shaking,	 and	 then	 cooled	 to	 room	
temperature.	 Then,	 3‐4	 drops	 of	 a	 freshly	 prepared	 sodium	
azide	solution	(2.5%	w:v)	was	added	to	remove	excess	KMnO4	
and	heated	gently	with	the	further	addition	of	2‐3	mL	of	water,	
if	necessary,	for	5	min	to	drive	off	the	excess	azide	solution	and	
cooled	 to	 room	 temperature.	 The	 reaction	 mixture	 was	
neutralized	with	 dilute	 NH4OH	 and	 transferred	 quantitatively	
into	 a	 10	 mL	 volumetric	 flask.	 Then	 the	 total	 iron(III+II)	
content	 was	 determined	 according	 to	 the	 general	 procedure	
with	the	help	of	the	calibration	graph.		

An	 equal	 aliquot	 of	 the	 above	 iron(III	 +	 II)	 mixture	 was	
taken	 into	 a	 25	 mL	 beaker.	 One	 ml	 of	 0.01%	 (w:v)	 1,10‐
phenanthroline	was	added	to	mask	iron(II)	and	neutralize	with	
dilute	NH4OH.	After,	the	content	of	the	beaker	was	transferred	
into	 a	 10ml	 volumetric	 flask	 and	 its	 iron	 (III)	 content	 was	
determined	 according	 to	 the	 general	 procedure.	 The	 iron	
concentration	was	calculated	in	mg/L	or	μg/L	with	the	aid	of	a	
calibration	 graph.	 This	 gives	 a	 measure	 of	 iron	 originally	
present	 as	 iron(III)	 in	 the	mixture.	 The	 value	 of	 the	 iron	 (II)	
concentration	was	calculated	by	subtracting	the	concentration	
of	 iron(III)	 from	 the	 corresponding	 total	 iron	 concentration.	
The	results	were	found	to	be	highly	reproducible.	The	results	of	
a	set	of	determination	are	given	in	Table	11.	
	
4.	Conclusions	
	

In	the	present	work,	a	new,	simple,	sensitive,	selective	and	
inexpensive	 spectrophotometric	method	with	 the	Fe(III)‐tiron	

complex	was	 developed	 for	 the	 determination	 of	 iron	 in	 real,	
environmental,	 biological,	 pharmaceutical,	 food	 and	 soil	
samples	for	continuous	monitoring	to	establish	the	trace	levels	
of	iron	in	different	sample	matrices.	The	similar	new,	sensitive	
and	selective	methods	[33‐39]	were	reported	by	the	author.		

Compared	with	the	other	methods	in	the	literature	[13‐18]	
the	 proposed	 method	 has	 several	 remarkable	 analytical	
characteristics:	

i) This	 method	 was	 developed	 in	 completely	 aqueous	
media,	 so	 toxic	 and	 carcinogenic	 organic	 solvents	
were	totally	avoided.	

ii) The	proposed	method	 is	highly	 sensitive	with	molar	
absorptivity	of	the	complex	of	6×105	L/mol.cm.	Thus	
amount	 of	 ng/g	 of	 iron	 can	 be	 determined	 without	
pre‐concentration.	

iii) The	 proposed	 method	 is	 very	 simple,	 rapid	 and	
stable.	 The	 reaction	 of	 iron(III)with	 tiron	 is	
completed	rapidly	in	aqueous	medium	within1min	at	
room	 temperature	 and	 offer	 the	 advantage	 of	 high	
complex	stability	(24	h).	

iv) The	 method	 has	 added	 advantages	 of	 determining	
individual	amounts	of	Fe(III)	and	Fe(II).	

v) With	 suitable	 masking	 agents,	 the	 reaction	 can	 be	
made	highly	selective.	

The	proposed	method	using	 tiron	 in	 aqueous	solution	not	
only	 is	 one	 of	 the	 most	 sensitive	 methods	 for	 the	 trace	
determination	 of	 iron	 but	 also	 is	 excellent	 in	 terms	 of	
sensitivity	 and	 simplicity.	 Therefore,	 this	 method	 will	 be	
successfully	applied	to	the	monitoring	of	trace	amounts	of	iron	
in	 real,	 environmental,	 industrial	 effluents,	 biological,	 food,	
pharmaceutical	and	soil	samples.	
	
Acknowledgements	
	

We	 are	 highly	 grateful	 to	 the	 authorities	 of	 Chittagong	
Medical	College	Hospital	for	supplying	Biological	samples.	
	
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